Lackland Air Force Base,3 Te-xas. Received 21 April 1994/Returned for modification 7 June 1994/Accepted 29 June 1994. To investigate the immune response ...
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INFECTION AND IMMUNITY, Sept. 1994, p. 3980-3983 0019-9567/94/$04.00+0 Copyright X 1994, American Society for Microbiology
Cytokine Induction in Human Coccidioidomycosis DAVID P. DOOLEY,1'2 REBECCA A. COX,'* KATHLEEN L. HESTILOW,' MATTHEW J. DOLAN,3 AND D. MITCHELL MAGEE' Department of Research Immunology, Texas Center for Infectious Disease, San Antonio,1 Infectious Disease Service, Brooke Army Medical Center, Ft. Sam Houston,2 and Wilford Hall Medical Center, Lackland Air Force Base,3 Te-xas Received 21 April 1994/Returned for modification 7 June 1994/Accepted 29 June 1994
To investigate the immune response to human infection with the fungus Coccidioides immitis, we measured cytokine production from peripheral blood mononuclear cells (PBMC) and plastic-adherent monocytes/ macrophages (M+) isolated from healthy subjects who were skin test positive to spherulin, healthy subjects who were skin test negative, and patients with active coccidioidomycosis. PBMC and M4 from all these donor groups secreted increased levels of tumor necrosis factor alpha, interleukin-1o, and interleukin-6 in response to stimulation with formalin-killed spherules (FKS), as measured by enzyme-linked immunosorbent assays. Viable C. immitis spherules also stimulated PBMC and M4 from healthy subjects and patients to secrete tumor necrosis factor alpha, interleukin-1o, and interleukin-6, although at levels lower than those induced by FKS. The production of these acute inflammatory cytokines may contribute to the immunopathogenesis of active coccidioidomycosis and could account for the toxicity of the FKS vaccine in humans.
granulomas (3, 14). It seems reasonable, then, to hypothesize that the induction in vivo of these cytokines may contribute to the pathogenesis of disease with C. immitis. The induction of TNF-a could also augment host defense in coccidioidomycosis, as suggested by the finding that TNF-a can synergize with gamma interferon to potentiate the anticoccidioidal effects of human monocytes (9) and murine macrophages (8). Evidence for a role for cytokines in the host response to C. immitis is derived from previous studies which showed that viable or formalin-killed spherules (FKS) induce murine peritoneal macrophages to secrete TNF-a. in vitro (28) and that actively infected BALB/c and DBAI2 mice secrete increased levels of TNF-a, IL-1p, and IL-6 (20). Ampel has recently extended these results to show that autoclaved spherules and arthroconidia of C. immitis induce the production of TNF-a by adherent mononuclear cells from healthy human donors (4). In this investigation, we examined the production of TNF-a, IL-1,, and IL-6 by human peripheral blood mononuclear cells (PBMC) and adherent monocytes (M+) in response to stimulation with FKS or viable spherules.
Coccidioidomycosis is a granulomatous infection caused by the fungus Coccidioides immitis, a soil saprophyte endemic to the southwestern United States. One hundred thousand new infections, with 70 deaths, occur in the area of endemicity every year (11, lla). Despite recent advances in antifungal therapy, disseminated disease and death still threaten certain patient populations, in particular, pregnant women, patients with AIDS, and certain racial groups (5). The inability to contain infection with C. immitis may be attributable to differences in the immune response to the organism (10, 11, lla). Therefore, investigations into the immunopathogenesis of this disease may direct the development of improved therapeutic or vaccine strategies. Human infection with C. immitis can be associated with a chronic wasting state similar to that seen in patients with tuberculosis or malignancies. A chronic, inexorably progressive granulomatous destruction of lung tissue may also occur, as may a syndrome resembling overwhelming pneumonia and septic shock (11, lla, 19). The immunopathogenesis of the wasting state, of the granulomatous destruction of lung tissue, and of the shock-like syndrome in this disease has not been well characterized. Tumor necrosis factor alpha (TNF-oa), interleukin-l,B (IL1 i), and IL-6 are cytokines produced predominantly by mononuclear phagocytes and have been demonstrated, physiologically, to mediate the normal immune response in humans (2, 18). These cytokines may, however, be responsible for the pathogenesis of multiple adverse effects which characterize such diverse disease states as septic shock (2, 18, 29, 30, 31) and meningitis (12) or the production of acute-phase reactants during acute illnesses (2, 18). TNF-ao has also been implicated in the induction of chronic wasting as a manifestation of more-indolent diseases such as malignancies (1, 6), human immunodeficiency virus infection (17, 21, 26), and tuberculosis (7, 23). Recent evidence also suggests that TNF-a may be essential for the effective development and function of tissue
MATERUILS AND METHODS Subjects. Sixteen patients with coccidioidomycosis were recruited from the wards and clinics of the Texas Center for Infectious Disease, Brooke Army Medical Center, Wilford Hall Medical Center, and Audie L. Murphy Memorial Veterans Hospital. Seven patients had disease limited to the lungs, while nine patients had disseminated disease defined as involvement of at least one extrapulmonary site. All patients had clinical evidence of active disease at the time of study. Twentyeight healthy subjects were recruited from hospital personnel at these same institutions. Of these, 15 were skin test positive (.5-mm induration at 24 or 48 h) to spherulin (usual test strength; Berkeley Biologicals, Berkeley, Calif.). The study was approved by the Institutional Review Boards at each institution and informed consent was obtained from each donor. Antigens. C. immitis spherules (Silveira strain) were grown for 5 days at 40°C on a gyratory shaker at 120 rpm in a modified liquid Converse medium as previously described (28). The cells were harvested by centrifugation, washed in pyrogen-free
* Corresponding author. Mailing address: Texas Center for Infectious Disease, San Antonio, TX 78223-3597. Phone: (210) 534-8857. Fax: (210) 532-7791.
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water, and then killed by the addition of formalin at a final concentration of 1%. After overnight incubation at 4°C, the FKS were harvested by centrifugation, washed in pyrogen-free water, and then lyophilized. Precautions were taken throughout the preceding steps to minimize the risk of contamination with endotoxin, and when assayed by a chromogenic Limulus amebocyte lysate test (M.A. Bioproducts, Inc., Walkersville, Md.), there was less than 11 pg of endotoxin per 10 ,ug of FKS. Lipopolysaccharide (LPS) from Escherichia coli serotype 055:B5 was obtained from Sigma Chemical Company (St. Louis, Mo.). Cytokine induction. Venous blood was collected in heparinized tubes (final concentration of 20 U of heparin per ml of blood), and the mononuclear cells were isolated by gradient centrifugation on Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, N.J.). PBMC were washed twice in RPMI 1640 medium and resuspended in medium containing 5% human AB serum (Sigma), penicillin (100 U/ml), streptomycin (100 mg/ml), and L-glutamine (2 mM) (hereafter designated complete RPMI). The cells were enumerated by hemacytometer counts and suspended to 5 x 106/ml of complete RPMI, and 0.1-ml portions of the suspension were dispensed into 96-well chambers. The monocyte composition of PBMC was comparable among the study groups, with a mean of 28.3%, as determined by hemacytometer counts. For monocyte isolation, the PBMC were resuspended to 2 x 106 monocytes per ml, and 0.1-ml portions were dispensed into 96-well chambers. Following incubation for 2 h at 37°C, the wells were gently washed three times with medium to remove nonadherent cells, and then the cells were reconstituted with 0.1 ml of complete RPMI per well. At least 95% of the adherent cells were monocytes/macrophages as judged by nonspecific esterase staining (15). Viability of PBMC and M( cultures was consistently greater than 90% by trypan blue dye exclusion, performed at the time the cultures were terminated. Cells were cultured in 0.2 ml of medium alone (to assess constitutive cytokine production) or stimulated with 10 jig of FKS, 104 viable spherules, or 1 ,ug of LPS, each diluted in complete RPMI. Preliminary studies established these concentrations as optimal for stimulation of cytokine production. After 18 to 24 h of incubation at 37°C under 5% C02, supernatants were collected and stored at -70°C until assayed. Cytokine assays. Supernatants were assayed for TNF-a by an enzyme-linked immunosorbent assay (ELISA) obtained from Endogen, Inc. (Boston, Mass.). IL-1, and IL-6 were assayed with ELISA kits purchased from Cistron Biotechnology (Pine Brook, N.J.). The results are expressed as the mean cytokine levels (picograms per milliliter) ± standard errors. Statistical analysis. Differences in the cytokine responses between the study groups were analyzed by the nonparametric Mann-Whitney U statistic. Comparisons of cytokine production by PBMC and MX were made by the Wilcoxon signed rank test.
RESULTS Cytokine production by PBMC. The production of TNF-a, IL-1p, and IL-6 by PBMC incubated for 18 to 24 h with FKS (10 ,ug per well) is shown in Fig. 1. Cells from all three study groups secreted significant levels of each cytokine in response to FKS (P < 0.0001 compared with unstimulated cultures). Although PBMC from healthy, skin test-negative subjects secreted lower levels of IL-6 than did healthy, skin test-positive persons and patients with coccidioidomycosis, the difference was not significant (P > 0.05). PBMC from the three donor groups also produced comparable levels of TNF-ot, IL-1p, and
CYTOKINE INDUCTION IN COCCIDIOIDOMYCOSIS IL-6
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FIG. 1. Production of TNF-ao, IL-1p, and IL-6 by PBMC (5 x 105 per well) following 18 to 24 h of stimulation with FKS (10 jig per well). Data were obtained from .10 subjects per group.
IL-6 in response to stimulation with LPS (results not shown). No endogenous cytokine production from unstimulated cells was observed. To exclude the possibility that cytokine production in response to FKS was attributable to an effect of formalin on antigenic determinants, PBMC cultures were also stimulated with viable C. immitis spherules. As shown in Fig. 2, PBMC from patients with active disease showed a dose-dependent production of TNF-a in response to viable spherules, at levels comparable to those produced by PBMC from healthy subjects who were skin test positive or negative to spherulin. IL-10 and IL-6 were also produced in response to viable spherules (results not shown). The induction of cytokines by viable spherules, which were devoid of detectable levels of LPS, argues against the possibility the cytokine responses induced by FKS were attributable to contaminating LPS. This interpretation is further supported by the finding that stimulation of PBMC with 11 pg of LPS, i.e., the highest level of contamination detected in FKS preparations, elicited less than 10% of the level of TNF-ot induced by the viable or killed
spherules. Healthy Subjects 140D4
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FIG. 2. TNF-a production from PBMC (5 x 105 per well) isolated from healthy subjects (skin test positive and negative) or patients with active coccidioidomycosis after 18 to 24 h of stimulation with FKS (0), at a concentration of 1 or 10 ,ug per well, or viable spherules (0) diluted to 103 or 104 CFU per well. Data were obtained from four to nine subjects per group.
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FIG. 3. Production of TNF-a, IL-1,3, and IL-6 by M4f (2 x 105 per well) following 18 to 24 h of stimulation with FKS (10 jig per well). Data were obtained from .10 subjects per group.
Cytokine production by M+. Although TNF-a, IL-1,B, and IL-6 are produced principally by M+, other effector cell populations may produce or modulate the production of these cytokines (2, 18). To assess cytokine secretion by enriched blood M+, PBMC from at least 10 subjects per donor group were fractionated by plastic adherence, and the adherent cells were stimulated with FKS (10 ,ug per well). The results are depicted in Fig. 3. Enriched M4) from the three donor groups secreted comparable levels of TNF-at and IL-1,, whereas production of IL-6 by M41 from healthy skin test-negative persons was decreased compared with that of healthy skin test-positive subjects or patients with active coccidioidomycosis (P < 0.05). It is noteworthy that the levels of cytokines produced by enriched M4 in response to stimulation with FKS were significantly decreased compared with the levels secreted by FKSstimulated PBMC (P < 0.0025) (compare Fig. 3 and 1). In contrast, cytokine production by LPS-stimulated MO was comparable to that of PBMC (P > 0.05). DISCUSSION The results of this study establish that viable or killed spherules of C. immitis induce PBMC and M4 from healthy subjects and patients with active coccidioidomycosis to secrete TNF-a, IL-1p, and IL-6. No correlation between PBMC cytokine responses to C. immitis and the ability of the patients with coccidioidomycosis to control or limit the extent of disease was observed. Rather, PBMC and M4 from patients secreted TNF-ao, IL-1p, and IL-6 at levels comparable to those produced by healthy, skin test-positive subjects. These results do not exclude the possibility, however, that differences may occur in the responses of cells at the site of infection, e.g., lung M4+. Investigations of cytokine production by compartmentalized cells have, in other diseases, revealed differences not detectable by using PBMC (13, 25). Ampel recently examined TNF-ao production by peripheral blood-derived adherent cells from healthy spherulin skin testpositive and -negative donors in response to stimulation with heat-killed spherules and arthroconidia (4). He reported that TNF-a production was increased in cells from healthy, skin test-positive persons when the supernatants were assayed by the L929 cytotoxicity bioassay. No differences were evident, however, when the supernatants were assayed by ELISA, which, unlike the bioassay, is specific for TNF-a (22). Significantly higher levels of cytokines were produced by PBMC than by M+, even when the cytokine levels were calculated on the basis of the number of M4 per well.
LPS-induced cytokine production, however, was comparable between M41 and PBMC. The increased production by PBMC suggests that other cells may have contributed to the production of these cytokines, either by producing the cytokines themselves or by potentiating the cytokine responses of monocytes (2, 18). Alternatively, the decreased production of cytokines in monocyte cultures could be attributable to a loss of viability or the number of cells as a result of the plastic adherence procedure used to enrich for monocytes, although these problems were not evident by microscopic analyses of the monolayers, by viability staining with trypan blue, or by the enumeration of nonadherent cells. The possibility that increased production of TNF-ot, IL-1,, and IL-6 by PBMC, compared with that by M4+, is attributable to the immunomodulatory effects of nonmonocytic cells could have important implications for host defense against C. immitis and requires further study. Cytokine production during infection with C. immitis may be responsible for several clinical features of the disease. Local production of cytokines may underlie the progressive fibrosis that characterizes chronic pulmonary coccidioidomycosis (16, 27). Systemic overproduction of TNF-a, IL-1p, and IL-6 may be responsible for the constitutional symptoms of fever, anorexia, and weight loss that are commonly associated with this disease (2, 18, 29) and could account for the recently described sepsis-like syndrome observed in patients with acute coccidioidomycosis (19). The induction of these cytokines could also explain the pronounced toxic manifestations associated with the FKS vaccine (32). Support for this hypothesis is derived from the observation that mice injected intraperitoneally with FKS manifest an immediate shock-like response characterized by ruffled fur and the development of an acute diarrhea (unpublished data). This toxic reaction resembles that induced by endotoxin (LPS) and is accompanied by increased serum levels of TNF-a, IL-11, and IL-6 (24; unpublished data). If the component(s) of FKS that induces the production of cytokines is distinct from that which affords protection, recombinant DNA technology could be used to produce a peptide vaccine that expresses only the immunoprotective epitopes. ACKNOWLEDGMENTS We thank Barbara Seaworth, Ken McAllister, Kay Sharkey, Alan Barbour, and the staffs of the Texas Center for Infectious Disease, Brooke Army Medical Center, and Wilford Hall Medical Center for the referral of patients for this study. We express our appreciation to the healthy subjects and patients who volunteered for this study. This work was supported, in part, by Brooke Army Medical Center protocol no. C-93-18 and Wilford Hall Medical Center protocol no. 89-119.
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